PET Imaging of Differentiated Thyroid Cancer with TSHR-Targeted [89Zr]Zr-TR1402.

Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [89Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [89Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89Zr (t1/2 = 78.4 h, ß+ = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [89Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [89Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T (P < 0.0001) and FTC133 (P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T (P < 0.0001) and TSHR+ FTC133 (P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [89Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference (P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference (P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [89Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

Somatostatin Receptor Type 2 and Thyroid-Stimulating Hormone Receptor Expression in Oncocytic Thyroid Neoplasms: Implications for Prognosis and Treatment.

Somatostatin receptor type 2 (SSTR2) and thyroid-stimulating hormone receptor (TSHR) display variable expression in primary thyroid tumors and have been implicated as theranostic targets. This study was designed to explore the differential expression of SSTR2 and TSHR in oncocytic (Hurthle cell) carcinoma (OC) vs oncocytic adenoma (OA). We performed a retrospective review for oncocytic neoplasms treated at our institution from 2012 to 2019. Formalin-fixed paraffin-embedded tissue blocks were used for tissue microarray construction. Tissue microarray blocks were cut into 5-µm sections and stained with anti-SSTR2 and anti-TSHR antibodies. Immunostains were analyzed by 3 independent pathologists. χ2 and logistic regression analysis were used to analyze clinical and pathologic variables. Sixty-seven specimens were analyzed with 15 OA and 52 OC. The mean age was 57 years, 61.2% were women, and 70% were White. SSTR2 positivity was noted in 2 OA (13%) and 15 OC (28%; 10 primary, 4 recurrent, and 1 metastatic) (P = .22). TSHR positivity was noted in 11 OA (73%) and 32 OC (62%; 31 primary and 1 metastatic) (P = .40). Those who presented with or developed clinical recurrence/metastasis were more likely to be SSTR2-positive (50% vs 21%; P = .04) and TSHR-negative (64.3% vs 28.9%; P = .02) than primary OC patients. Widely invasive OC was more likely to be SSTR2-positive compared to all other OC subtypes (minimally invasive and angioinvasive) (P = .003). For all patients with OC, TSHR positivity was inversely correlated with SSTR2 positivity (odds ratio, 0.12; CI, 0.03-0.43; P = .006). This relationship was not seen in the patients with OA (odds ratio, 0.30; CI, 0.01-9.14; P = .440). Our results show that recurrent/metastatic OC was more likely to be SSTR2-positive and TSHR-negative than primary OC. Patients with OC displayed a significant inverse relationship between SSTR2 and TSHR expression that was not seen in patients with OA. This may be a key relationship that can be used to prognosticate and treat OCs.

Synthetic Makaluvamine Analogs Decrease c-Kit Expression and Are Cytotoxic to Neuroendocrine Tumor Cells.

In an effort to discover viable systemic chemotherapeutic agents for neuroendocrine tumors (NETs), we screened a small library of 18 drug-like compounds obtained from the Velu lab against pulmonary (H727) and thyroid (MZ-CRC-1 and TT) neuroendocrine tumor-derived cell lines. Two potent lead compounds (DHN-II-84 and DHN-III-14) identified from this screening were found to be analogs of the natural product makaluvamine. We further characterized the antitumor activities of these two compounds using pulmonary (H727), thyroid (MZ-CRC-1) and pancreatic (BON) neuroendocrine tumor cell lines. Flow cytometry showed a dose-dependent increase in apoptosis in all cell lines. Induction of apoptosis with these compounds was also supported by the decrease in myeloid cell leukemia-1 (MCL-1) and X-chromosome linked inhibitor of apoptosis (XIAP) detected by Western blot. Compound treatment decreased NET markers chromogranin A (CgA) and achaete-scute homolog 1 (ASCL1) in a dose-dependent manner. Moreover, the gene expression analysis showed that the compound treatment reduced c-Kit proto-oncogene expression in the NET cell lines. Induction of apoptosis could also have been caused by the inhibition of c-Kit expression, in addition to the known mechanisms such as damage of DNA by topoisomerase II inhibition for this class of compounds. In summary, makaluvamine analogs DHN-II-84 and DHN-III-14 induced apoptosis, decreased neuroendocrine tumor markers, and showed promising antitumor activity in pulmonary, thyroid, and pancreatic NET cell lines, and hold potential to be developed as an effective treatment to combat neuroendocrine tumors.

The Role of Notch3 in Cancer.

The Notch family is a highly conserved gene group that regulates cell-cell interaction, embryogenesis, and tissue commitment. This review article focuses on the third Notch family subtype, Notch3. Regulation via Notch3 signaling was first implicated in vasculogenesis. However, more recent findings suggest that Notch3 signaling may play an important role in oncogenesis, tumor maintenance, and resistance to chemotherapy. Its role is mainly oncogenic, although in some cancers it appears to be tumor suppressive. Despite the wealth of published literature, it remains relatively underexplored and requires further research to shed more light on its role in cancer development, determine its tissue-specific function, and elaborate novel treatment strategies. Herein we summarize the role of Notch3 in cancer, possible mechanisms of its action, and current cancer treatment strategies targeting Notch3 signaling. IMPLICATIONS FOR PRACTICE: The Notch family is a highly conserved gene group that regulates cell-cell interaction, embryogenesis, and tissue commitment. This review summarizes the existing data on the third subtype of the Notch family, Notch3. The role of Notch3 in different types of cancers is discussed, as well as implications of its modification and new strategies to affect Notch3 signaling activity.

Extracellular vesicles as carriers of microRNA, proteins and lipids in tumor microenvironment.

In recent years, the knowledge about the control of tumor microenvironment has increased and emerged as an important player in tumorigenesis. The role of normal stromal cells in the tumor initiation and progression has brought our vision in to the forefront of cell-to-cell communication. In this review, we focus on the mechanism of communication between stromal and tumor cells, which is based on the exchange of extracellular vesicles (EVs). We describe several, evergrowing, pieces of evidence that EVs transfer messages through their miRNA, lipid, protein and nucleic acid contents. A better understanding of this sophisticated method of communication between normal cancer cells may lead to developing novel approaches for personalized diagnostics and therapeutics.

Thyroid-stimulating hormone receptor (TSHR) as a target for imaging differentiated thyroid cancer.

BACKGROUND: Of the half a million cases of thyroid cancer diagnosed annually, 95% are differentiated thyroid cancers. Although clinical guidelines recommend surgical resection followed by radioactive iodine ablation, loss of sodium-iodine symporter expression causes up to 20% of differentiated thyroid cancers to become radioactive iodine refractory. For patients with radioactive iodine refractory disease, there is an urgent need for new diagnostic and therapeutic approaches. We evaluated the thyroid-stimulating hormone receptor as a potential target for imaging of differentiated thyroid cancer. METHODS: We immunostained tissue microarrays containing 52 Hurthle cell carcinomas to confirm thyroid-stimulating hormone receptor expression. We radiolabeled chelator deferoxamine conjugated to recombinant human thyroid-stimulating hormone analog superagonist TR1402 with 89Zr (t1/2 = 78.4 h, ß+ =22.7%) to produce [89Zr]Zr-TR1402. We performed in vitro uptake assays in high-thyroid-stimulating hormone receptor and low-thyroid-stimulating hormone receptor-expressing THJ529T and FTC133 thyroid cancer cell lines. We performed in vivo positron emission tomography/computed tomography and biodistribution studies in male athymic nude mice bearing thyroid-stimulating hormone receptor-positive THJ529T tumors. RESULTS: Immunohistochemical analysis revealed 62% of patients (27 primary and 5 recurrent) were thyroid-stimulating hormone receptor membranous immunostain positive. In vitro uptake of 1nM [89Zr]Zr-TR1402 was 38 ± 17% bound/mg in thyroid-stimulating hormone receptor-positive THJ529T thyroid cancer cell lines compared to 3.2 ± 0.5 in the low-expressing cell line (P < .01), with a similar difference seen in FTC133 cell lines (P < .0001). In vivo and biodistribution studies showed uptake of [89Zr]Zr-TR1402 in thyroid-stimulating hormone receptor-expressing tumors, with a mean percentage of injected dose/g of 1.9 ± 0.4 at 3 days post-injection. CONCLUSION: Our observation of thyroid-stimulating hormone receptor expression in tissue microarrays and [89Zr]Zr-TR1402 accumulation in thyroid-stimulating hormone receptor-positive thyroid cancer cells and tumors suggests thyroid-stimulating hormone receptor is a promising target for imaging of differentiated thyroid cancer.

Voltage-Gated Sodium Channel NaV1.7 Inhibitors with Potent Anticancer Activities in Medullary Thyroid Cancer Cells.

Our results from quantitative RT-PCR, Western blotting, immunohistochemistry, and the tissue microarray of medullary thyroid cancer (MTC) cell lines and patient specimens confirm that VGSC subtype NaV1.7 is uniquely expressed in aggressive MTC and not expressed in normal thyroid cells and tissues. We establish the druggability of NaV1.7 in MTC by identifying a novel inhibitor (SV188) and investigate its mode of binding and ability to inhibit INa current in NaV1.7. The whole-cell patch-clamp studies of the SV188 in the NaV1.7 channels expressed in HEK-293 cells show that SV188 inhibited the INa current in NaV1.7 with an IC50 value of 3.6 µM by a voltage- and use-dependent blockade mechanism, and the maximum inhibitory effect is observed when the channel is open. SV188 inhibited the viability of MTC cell lines, MZ-CRC-1 and TT, with IC50 values of 8.47 µM and 9.32 µM, respectively, and significantly inhibited the invasion of MZ-CRC-1 cells by 35% and 52% at 3 µM and 6 µM, respectively. In contrast, SV188 had no effect on the invasion of TT cells derived from primary tumor, which have lower basal expression of NaV1.7. In addition, SV188 at 3 µM significantly inhibited the migration of MZ-CRC-1 and TT cells by 27% and 57%, respectively.

Novel Non-Cyclooxygenase Inhibitory Derivative of Sulindac Inhibits Breast Cancer Cell Growth In Vitro and Reduces Mammary Tumorigenesis in Rats.

The nonsteroidal anti-inflammatory drug (NSAID) sulindac demonstrates attractive anticancer activity, but the toxicity resulting from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins precludes its long-term, high dose use in the clinic for cancer prevention or treatment. While inflammation is a known tumorigenic driver, evidence suggests that sulindac's antineoplastic activity is partially or fully independent of its COX inhibitory activity. One COX-independent target proposed for sulindac is cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) isozymes. Sulindac metabolites, i.e., sulfide and sulfone, inhibit cGMP PDE enzymatic activity at concentrations comparable with those associated with cancer cell growth inhibitory activity. Additionally, the cGMP PDE isozymes PDE5 and PDE10 are overexpressed during the early stages of carcinogenesis and appear essential for cancer cell proliferation and survival based on gene silencing experiments. Here, we describe a novel amide derivative of sulindac, sulindac sulfide amide (SSA), which was rationally designed to eliminate COX-inhibitory activity while enhancing cGMP PDE inhibitory activity. SSA was 68-fold and 10-fold less potent than sulindac sulfide (SS) in inhibiting COX-1 and COX-2, respectively, but 10-fold more potent in inhibiting growth and inducing apoptosis in breast cancer cells. The pro-apoptotic activity of SSA was associated with inhibition of cGMP PDE activity, elevation of intracellular cGMP levels, and activation of cGMP-dependent protein kinase (PKG) signaling, as well as the inhibition of ß-catenin/Tcf transcriptional activity. SSA displayed promising in vivo anticancer activity, resulting in a 57% reduction in the incidence and a 62% reduction in the multiplicity of tumors in the N-methyl-N-nitrosourea (MNU)-induced model of breast carcinogenesis. These findings provide strong evidence for cGMP/PKG signaling as a target for breast cancer prevention or treatment and the COX-independent anticancer properties of sulindac. Furthermore, this study validates the approach of optimizing off-target effects by reducing the COX-inhibitory activity of sulindac for future targeted drug discovery efforts to enhance both safety and efficacy.

Ex Vivo Modeling of Human Neuroendocrine Tumors in Tissue Surrogates.

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community's ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo.

Anti-SSTR2 antibody-drug conjugate for neuroendocrine tumor therapy.

Neuroendocrine (NE) tumors include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio-therapies have marginal curative benefits. The goal of this study was to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). First, we confirmed that somatostatin receptor 2 (SSTR2) is an ideal cancer cell surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and three NET cell lines. Then, we developed a new monoclonal antibody (mAb, IgG1, and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells. Finally, the ADC was evaluated in vivo using a NET xenograft mouse model to assess cancer-specific targeting, tolerated dosage, pharmacokinetics, and antitumor efficacy. The anti-SSTR2 ADC exclusively targeted and killed NET cells with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 ADC has a high-therapeutic potential for NET therapy.

Non-toxic fragment of botulinum neurotoxin type A and monomethyl auristatin E conjugate for targeted therapy for neuroendocrine tumors.

Surgical resection is the only cure for neuroendocrine tumors (NETs). However, widespread metastases have already occured by the time of initial diagnosis in many cases making complete surgical removal impossible. We developed a recombinant heavy-chain receptor binding domain (rHCR) of botulinum neurotoxin type A that can specifically target synaptic vesicle 2 (SV2), a surface receptor abundantly expressed in multiple neuroendocrine tumors. Expression of neuroendocrine differentiation markers chromogranin A (CgA) and achaete-scute complex 1 (ASCL1) were signficantly reduced when treated with rHCR. rHCR conjugated to the antimitotic agent monomethyl auristatin E (MMAE) significantly suppressed proliferation of pancreatic carcinoid (BON) and medullary thyroid cancer cells (MZ) at concentrations of 500 and 300 nM respectively, while no growth suppression was observed in pulmonary fibroblasts and cortical neuron control cell lines. In vivo, rHCR-MMAE significantly reduced tumor volume in mouse xenografts with no observed adverse effects. These data suggest recombinant HCR (rHCR) of BoNT/A preferentially targets neuroendocrine cancer without the neurotoxicity of the full BoNT/A and that SV2 is a specific and promising target for delivering drugs to neuroendocrine tumors.

Overexpression of somatostatin receptor type 2 in neuroendocrine tumors for improved Ga68-DOTATATE imaging and treatment.

BACKGROUND: Neuroendocrine tumors are found throughout the body, including the pancreas. These tumors are phenotypically and genetically heterogeneous and can be difficult to accurately image using current imaging standards. However, positron emission tomography/computed tomography with radiolabeled somatostatin analogs has shown clinical success because many neuroendocrine tumors overexpress somatostatin receptor subtype 2. Unfortunately, patients with poorly differentiated neuroendocrine tumors often have a diminished level of somatostatin receptor subtype 2. We found that histone deacetylase inhibitors can upregulate the functional expression of somatostatin receptor subtype 2. METHODS: We evaluated the effect of histone deacetylase inhibitors on somatostatin receptor subtype 2 expression at the mRNA and protein level in neuroendocrine tumor cell lines. The effect of histone deacetylase inhibitors on surface somatostatin receptor subtype 2 was also investigated by fluorescence-activated cell sorting analysis. Changes in somatostatin receptor subtype 2 expression in neuroendocrine tumor xenografts after treatment were imaged using Ga68-DOTATATE positron emission tomography/computed tomography. RESULTS: The functional increase of somatostatin receptor subtype 2 in neuroendocrine tumors after histone deacetylase inhibitor treatment was confirmed through in vitro experiments and small animal Ga68-DOTATATE positron emission tomography/computed tomography imaging. Histone deacetylase inhibitors increased somatostatin receptor subtype 2 transcription and protein expression in neuroendocrine tumor cell lines. Small animal Ga68-DOTATATE positron emission tomography/computed tomography imaging confirmed the enhancement of radiopeptide uptake after histone deacetylase inhibitor administration. CONCLUSION: This study demonstrates a new method to potentially improve imaging and treatments that target somatostatin receptor subtype 2 in neuroendocrine tumors.

A growth model of neuroendocrine tumor surrogates and the efficacy of a novel somatostatin-receptor-guided antibody-drug conjugate: Perspectives on clinical response?

BACKGROUND: As patient-derived xenografts and other preclinical models of neuroendocrine tumors for testing personalized therapeutics are lacking, we have developed a perfused, 3D bioreactor model to culture tumor surrogates from patient-derived neuroendocrine tumors. This work evaluates the duration of surrogate culture and surrogate response to a novel antibody-drug conjugate. METHODS: Twenty-seven patient-derived neuroendocrine tumors were cultured. Histologic sections of a pancreatic neuroendocrine tumor xenograft (BON-1) tumor were assessed for SSTR2 expression before tumor implantation into 2 bioreactors. One surrogate was treated with an antibody-drug conjugate composed of an anti-mitotic Monomethyl auristatin-E linked to a somatostatin receptor 2 antibody. Viability and therapeutic response were assessed by pre-imaging incubation with IR-783 and the RealTime-Glo AnnexinV Apoptosis and Necrosis Assay (Promega Corporation, Madison, WI) over 6 days. A primary human pancreatic neuroendocrine tumor was evaluated similarly. RESULTS: Mean surrogate growth duration was 34.8 days. Treated BON-1 surrogates exhibited less proliferation (1.2 vs 1.9-fold) and greater apoptosis (1.5 vs 1.1-fold) than controls, whereas treated patient-derived neuroendocrine tumor bioreactors exhibited greater degrees of apoptosis (13- vs 9-fold) and necrosis (2.5- vs 1.6-fold). CONCLUSION: Patient-derived neuroendocrine tumor surrogates can be cultured reliably within the bioreactor. This model can be used to evaluate the efficacy of antibody-guided chemotherapy ex vivo and may be useful for predicting clinical responses.

Pulmonary Carcinoid Surface Receptor Modulation Using Histone Deacetylase Inhibitors.

Pulmonary carcinoids are a type of neuroendocrine tumor (NET) accounting for 1-2% of lung cancer cases. Currently, Positron Emission Tomography (PET)/CT based on the radiolabeled sugar analogue [18F]-FDG is used to diagnose and stage pulmonary carcinoids, but is suboptimal due to low metabolic activity in these tumors. A new technique for pulmonary carcinoid imaging, using PET/CT with radiolabeled somatostatin analogs that specifically target somatostatin receptor subtype 2 (SSTR2), is becoming more standard, as many tumors overexpress SSTR2. However, pulmonary carcinoid patients with diminished SSTR2 expression are not eligible for this imaging or any type of SSTR2-specific treatment. We have found that histone deacetylase (HDAC) inhibitors can upregulate the expression of SSTR2 in pulmonary carcinoid cell lines. In this study, we used a non-cytotoxic dose of HDAC inhibitors to induce pulmonary carcinoid SSTR2 expression in which we confirmed in vitro and in vivo. A non-cytotoxic dose of the HDAC inhibitors: thailandepsin A (TDP-A), romidepsin (FK228), suberoylanilide hydroxamic acid (SAHA), AB3, and valproic acid (VPA) were administered to promote SSTR2 expression in pulmonary carcinoid cell lines and xenografts. This SSTR2 upregulation technique using HDAC inhibitors could enhance radiolabeled somatostatin analog-based imaging and the development of potential targeted treatments for pulmonary carcinoid patients with marginal or diminished SSTR2 expression.

Induced pluripotent stem cell-derived mesenchymal stem cells: A leap toward personalized therapies.

Mesenchymal Stem/stromal cell (MSCs) transplantation procedures have been used since the 1960's to treat leukemia and other diseases, but due to the risks involved only patients with life threatening illnesses were typically subjected to the transplantation procedure until the last decade. Recent advancements in transplantation techniques have made it more feasible to use it for non-life-threatening diseases. However, the potential uses for stem cells are still limited by their rarity, and, in the case of allogeneic transplants, graft-vs.-host complications. An evolving alternative to conventional stem cell therapies is induced pluripotent stem-cell derived mesenchymal stem/stromal cells (iPSC- MSCs), which have a multi-lineage potential comparable to conventionally acquired MSCs with the added benefit of being less immunoreactive. However there are still many hurdles left to be overcome before they can be used regularly for personalized therapies. This review will focus on recent advancements that have been made regarding the role MSCs play in tumor development and the potential uses iPSC-MSCs may have in future cancer treatment.

Mesenchymal Stem/Stromal Cells under Stress Increase Osteosarcoma Migration and Apoptosis Resistance via Extracellular Vesicle Mediated Communication.

Studies have shown that mesenchymal stem/stromal cells (MSCs) from bone marrow are involved in the growth and metastasis of solid tumors but the mechanism remains unclear in osteosarcoma (OS). Previous studies have raised the possibility that OS cells may receive support from associated MSCs in the nutrient deprived core of the tumors through the release of supportive macromolecules and growth factors either in vesicular or non-vesicular forms. In the present study, we used stressed mesenchymal stem cells (SD-MSCs), control MSCs and OS cells to examine the hypothesis that tumor-associated MSCs in nutrient deprived core provide pro-proliferative, anti-apoptotic, and metastatic support to nearby tumor cells. Assays to study of the effects of SD-MSC conditioned media revealed that OS cells maintained proliferation when compared to OS cells grown under serum-starved conditions alone. Furthermore, OS cells in MSCs and SD-MSC conditioned media were significantly resistant to apoptosis and an increased wound healing rate was observed in cells exposed to either conditioned media or EVs from MSCs and SD-MSCs. RT-PCR assays of OS cells incubated with extracellular vesicles (EVs) from SD-MSCs revealed microRNAs that could potentially target metabolism and metastasis associated genes as predicted by in silico algorithms, including monocarboxylate transporters, bone morphogenic receptor type 2, fibroblast growth factor 7, matrix metalloproteinase-1, and focal adhesion kinase-1. Changes in the expression levels of focal adhesion kinase, STK11 were confirmed by quantitative PCR assays. Together, these data indicate a tumor supportive role of MSCs in osteosarcoma growth that is strongly associated with the miRNA content of the EVs released from MSCs under conditions that mimic the nutrient deprived core of solid tumors.

Sulindac sulfide selectively increases sensitivity of ABCC1 expressing tumor cells to doxorubicin and glutathione depletion.

ATP-binding cassette (ABC) transporters ABCC1 (MRP1), ABCB1 (P-gp), and ABCG2 (BCRP) contribute to chemotherapy failure. The primary goals of this study were to characterize the efficacy and mechanism of the nonsteroidal anti-inflammatory drug (NSAID), sulindac sulfide, to reverse ABCC1 mediated resistance to chemotherapeutic drugs and to determine if sulindac sulfide can influence sensitivity to chemotherapeutic drugs independently of drug efflux. Cytotoxicity assays were performed to measure resistance of ABC-expressing cell lines to doxorubicin and other chemotherapeutic drugs. NSAIDs were tested for the ability to restore sensitivity to resistance selected tumor cell lines, as well as a large panel of standard tumor cell lines. Other experiments characterized the mechanism by which sulindac sulfide inhibits ABCC1 substrate and co-substrate (GSH) transport in isolated membrane vesicles and intact cells. Selective reversal of multi-drug resistance (MDR), decreased efflux of doxorubicin, and fluorescent substrates were demonstrated by sulindac sulfide and a related NSAID, indomethacin, in resistance selected and engineered cell lines expressing ABCC1, but not ABCB1 or ABCG2. Sulindac sulfide also inhibited transport of leukotriene C4 into membrane vesicles. Sulindac sulfide enhanced the sensitivity to doxorubicin in 24 of 47 tumor cell lines, including all melanoma lines tested (7-7). Sulindac sulfide also decreased intracellular GSH in ABCC1 expressing cells, while the glutathione synthesis inhibitor, BSO, selectively increased sensitivity to sulindac sulfide induced cytotoxicity. Sulindac sulfide potently and selectively reverses ABCC1-mediated MDR at clinically achievable concentrations. ABCC1 expressing tumors may be highly sensitive to the direct cytotoxicity of sulindac sulfide, and in combination with chemotherapeutic drugs that induce oxidative stress.

Extracellular Vesicles: Evolving Factors in Stem Cell Biology.

Stem cells are proposed to continuously secrete trophic factors that potentially serve as mediators of autocrine and paracrine activities, associated with reprogramming of the tumor microenvironment, tissue regeneration, and repair. Hitherto, significant efforts have been made to understand the level of underlying paracrine activities influenced by stem cell secreted trophic factors, as little is known about these interactions. Recent findings, however, elucidate this role by reporting the effects of stem cell derived extracellular vesicles (EVs) that mimic the phenotypes of the cells from which they originate. Exchange of genetic information utilizing persistent bidirectional communication mediated by stem cell-EVs could regulate stemness, self-renewal, and differentiation in stem cells and their subpopulations. This review therefore discusses stem cell-EVs as evolving communication factors in stem cell biology, focusing on how they regulate cell fates by inducing persistent and prolonged genetic reprogramming of resident cells in a paracrine fashion. In addition, we address the role of stem cell-secreted vesicles in shaping the tumor microenvironment and immunomodulation and in their ability to stimulate endogenous repair processes during tissue damage. Collectively, these functions ensure an enormous potential for future therapies.

A novel sulindac derivative that potently suppresses colon tumor cell growth by inhibiting cGMP phosphodiesterase and ß-catenin transcriptional activity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to inhibit tumor growth by a COX-independent mechanism, although alternative targets have not been well defined or used to develop improved drugs for cancer chemoprevention. Here, we characterize a novel sulindac derivative referred to as sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and induces the apoptosis of human colon tumor cells. The basis for this activity appears to involve cyclic guanosine 3',5',-monophosphate phosphodiesterase (cGMP PDE) inhibition as evident by its ability to inhibit cGMP hydrolysis in colon tumor cell lysates and purified cGMP-specific PDE5, increase intracellular cGMP levels, and activate cGMP-dependent protein kinase G at concentrations that suppress tumor cell growth. PDE5 was found to be essential for colon tumor cell growth as determined by siRNA knockdown studies, elevated in colon tumor cells as compared with normal colonocytes, and associated with the tumor selectivity of SBA. SBA activation of PKG may suppress the oncogenic activity of ß-catenin as evident by its ability to reduce ß-catenin nuclear levels, Tcf (T-cell factor) transcriptional activity, and survivin levels. These events preceded apoptosis induction and appear to result from a rapid elevation of intracellular cGMP levels following cGMP PDE inhibition. We conclude that PDE5 and possibly other cGMP degrading isozymes can be targeted to develop safer and more efficacious NSAID derivatives for colorectal cancer chemoprevention.

NSAIDs: Old Drugs Reveal New Anticancer Targets.

There is compelling evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 selective inhibitors have antineoplastic activity, but toxicity from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins limits their use for cancer chemoprevention. Previous studies as reviewed here suggest that the mechanism for their anticancer properties does not require COX inhibition, but instead involves an off-target effect. In support of this possibility, recent molecular modeling studies have shown that the NSAID sulindac can be chemically modified to selectively design out its COX-1 and COX-2 inhibitory activity. Unexpectedly, certain derivatives that were synthesized based on in silico modeling displayed increased potency to inhibit tumor cell growth. Other experiments have shown that sulindac can inhibit phosphodiesterase to increase intracellular cyclic GMP levels and that this activity is closely associated with its ability to selectively induce apoptosis of tumor cells. Together, these studies suggest that COX-independent mechanisms can be targeted to develop safer and more efficacious drugs for cancer chemoprevention.